Bottom Line:
In this respect they are remarkably similar to their non-malignant counterparts that orchestrate the intestinal lining.Moreover, the Wnt pathway, which carries activating mutations in virtually all colon cancers, is not as previously predicted constitutively active in adenomas and carcinomas, but still displays a heterogeneous activity pattern that determined stemness in all stages of disease.These data for the first time provide a comprehensive overview of Wnt and Notch-mediated signaling in the different stages of the adenoma-carcinoma sequence and demonstrates that these morphogenic pathways, despite mutations, remain crucial determinants of both architecture and hierarchy in normal and malignant intestinal tissue.

Background: Colon cancer stem cells are shown to be the self-renewing cells within a tumor that give rise to all lineages of more differentiated tumor cells. In this respect they are remarkably similar to their non-malignant counterparts that orchestrate the intestinal lining. This suggests that, despite the numerous genetic aberrations and morphological changes that have occurred during cancer initiation and progression, a remnant homeostatic regulation persists.

Findings: Using a number of human and mouse intestinal-derived organoid cultures from normal, adenoma and cancerous tissues, we show here that Notch signals coordinate self-renewal and lineage determination not only in normal, but also at the adenoma and carcinoma stage in both mice and humans. Moreover, the Wnt pathway, which carries activating mutations in virtually all colon cancers, is not as previously predicted constitutively active in adenomas and carcinomas, but still displays a heterogeneous activity pattern that determined stemness in all stages of disease.

Conclusion: These data for the first time provide a comprehensive overview of Wnt and Notch-mediated signaling in the different stages of the adenoma-carcinoma sequence and demonstrates that these morphogenic pathways, despite mutations, remain crucial determinants of both architecture and hierarchy in normal and malignant intestinal tissue.

Figure 2: The Notch pathway is analogously orchestrated throughout CRC progression. (A) Representative confocal microscopy images of MUC2 (green) in human cultures treated with 10 μM dibenzazepine (DBZ) show differentiation into the goblet cell-lineage as depicted by increased MUC2 expression (B) The number of goblet cells was quantified by counting the number of MUC2+ cells from 10 random fields at 63× magnification. Values shown are the percentages of MUC2+ cells relative to the total number of cells. (C) The increased MUC2 expression was further measured with qRT-PCR. MUC2 mRNA levels were normalized to GAPDH mRNA and expressed as fold induction compared with DMSO control. The graph represents the mean ± SD of three different experiments. (D) DBZ treatment results in reduced clonogenicity. The results depicted are representative from three independent experiments from human healthy colon organoid (N=2), adenoma (N=3) and adenocarcinoma (N=3). Scale bar: 50 μm. * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001 (t-test).

Mentions:
To address this, we first assessed the effect of pharmacological inhibition of the Notch pathway in the distinct cultures. As previously reported, dibenzazepine (DBZ) mediated Notch inhibition resulted in goblet cell differentiation in cultured mouse intestinal epithelium and adenoma (Additional file 2: Figure S2). Importantly, also treatment of human epithelial organoid cultures derived either from normal, adenoma or adenocarcinoma tissues with DBZ, caused a clear increase in goblet cells (Figure 2A-B) and a significant induction of MUC2 mRNA level (Figure 2C). This goblet cell-like differentiation appeared to be observed only in cultures already expressing low amounts of MUC2 (Additional file 3: Figure S3). More importantly, the clonogenic potential of all cultures was clearly decreased upon DBZ treatment, which is consistent with the loss of (cancer) stem cells (Figure 2D and Additional file 2: Figure S2), implying that Notch pathway-dependent self-renewal and cell fate decision are maintained at all stages of tumor development.

Figure 2: The Notch pathway is analogously orchestrated throughout CRC progression. (A) Representative confocal microscopy images of MUC2 (green) in human cultures treated with 10 μM dibenzazepine (DBZ) show differentiation into the goblet cell-lineage as depicted by increased MUC2 expression (B) The number of goblet cells was quantified by counting the number of MUC2+ cells from 10 random fields at 63× magnification. Values shown are the percentages of MUC2+ cells relative to the total number of cells. (C) The increased MUC2 expression was further measured with qRT-PCR. MUC2 mRNA levels were normalized to GAPDH mRNA and expressed as fold induction compared with DMSO control. The graph represents the mean ± SD of three different experiments. (D) DBZ treatment results in reduced clonogenicity. The results depicted are representative from three independent experiments from human healthy colon organoid (N=2), adenoma (N=3) and adenocarcinoma (N=3). Scale bar: 50 μm. * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001 (t-test).

Mentions:
To address this, we first assessed the effect of pharmacological inhibition of the Notch pathway in the distinct cultures. As previously reported, dibenzazepine (DBZ) mediated Notch inhibition resulted in goblet cell differentiation in cultured mouse intestinal epithelium and adenoma (Additional file 2: Figure S2). Importantly, also treatment of human epithelial organoid cultures derived either from normal, adenoma or adenocarcinoma tissues with DBZ, caused a clear increase in goblet cells (Figure 2A-B) and a significant induction of MUC2 mRNA level (Figure 2C). This goblet cell-like differentiation appeared to be observed only in cultures already expressing low amounts of MUC2 (Additional file 3: Figure S3). More importantly, the clonogenic potential of all cultures was clearly decreased upon DBZ treatment, which is consistent with the loss of (cancer) stem cells (Figure 2D and Additional file 2: Figure S2), implying that Notch pathway-dependent self-renewal and cell fate decision are maintained at all stages of tumor development.

Bottom Line:
In this respect they are remarkably similar to their non-malignant counterparts that orchestrate the intestinal lining.Moreover, the Wnt pathway, which carries activating mutations in virtually all colon cancers, is not as previously predicted constitutively active in adenomas and carcinomas, but still displays a heterogeneous activity pattern that determined stemness in all stages of disease.These data for the first time provide a comprehensive overview of Wnt and Notch-mediated signaling in the different stages of the adenoma-carcinoma sequence and demonstrates that these morphogenic pathways, despite mutations, remain crucial determinants of both architecture and hierarchy in normal and malignant intestinal tissue.

Background: Colon cancer stem cells are shown to be the self-renewing cells within a tumor that give rise to all lineages of more differentiated tumor cells. In this respect they are remarkably similar to their non-malignant counterparts that orchestrate the intestinal lining. This suggests that, despite the numerous genetic aberrations and morphological changes that have occurred during cancer initiation and progression, a remnant homeostatic regulation persists.

Findings: Using a number of human and mouse intestinal-derived organoid cultures from normal, adenoma and cancerous tissues, we show here that Notch signals coordinate self-renewal and lineage determination not only in normal, but also at the adenoma and carcinoma stage in both mice and humans. Moreover, the Wnt pathway, which carries activating mutations in virtually all colon cancers, is not as previously predicted constitutively active in adenomas and carcinomas, but still displays a heterogeneous activity pattern that determined stemness in all stages of disease.

Conclusion: These data for the first time provide a comprehensive overview of Wnt and Notch-mediated signaling in the different stages of the adenoma-carcinoma sequence and demonstrates that these morphogenic pathways, despite mutations, remain crucial determinants of both architecture and hierarchy in normal and malignant intestinal tissue.